Pharmaceutical compositions for reducing hyperlipidemia and platelet-aggregation

ABSTRACT

The new pharmaceutical compositions and processes are provided for reducing both hyperlipidemia and platelet-aggregation (PHP). 
     The fine-PHP is composed of following ingredients: Scoparone or Aurapten, Curcumin, Ferulic Acid, Yejuhua-flavonoid and additive ingredient--fish oil. The compositions are nontoxic.

This application is a division of application Ser. No. 905,554, filedSep. 8, 1986, now U.S. Pat. No. 4,842,859.

BACKGROUND OF THE INVENTION

The present invention relates to pharmaceutical compositions useful forreducing hyperlipidemia and platelet-aggregation. The bifunctionalpharmaceutical compositions are nontoxic.

Specifically, this invention provides new compositions of five majoractive ingredients.

1. Aurapten or scoparone is extracted from peels seeds or young plantsof Citrus aurantium L., Citrus spp., Poncirus trifoliata raf orArtemisia capillaris Thunb.

2. Curcumin is extracted from Curcuma longa L., Curcuma aromatica salisbor Curcuma zedoaria roscoe.

3. Ferulic acid is extracted from Levisticum officinale koch, Angelicasinensis Diels or Angelica spp.

4. Yejuhua-flavonoid is extracted from Matricaria chamomilla L, Tagetesminuta L, Tagetes patula L or Chrysanthemum indicum L.

The herbs: Citrus aurantium L, Citrus spp, Curcuma longa L, Levisticumofficinale koch, Angelica spp, Matricaria chamomilla L, Tagetes minuta Land Tagetes patula L are recognized by the U.S. Food and DrugAdministration (FDA) as safe for human consumption.

5. Additive ingredient: fish oil concentrate derived from the fish body(including scale of a fish).

DESCRIPTION OF THE PRIOR ART

Some drugs have been used for decreasing levels of cholesterol andtriglyceride: for example, Clofibrate, Gemfibrozil, Questran, Colestipoland Neomycin in particular, for decreasing cholesterol and triglyceridelevels in hypercholesterol and hypertriglyceridemia.

However, all the above as mentioned drugs have a certain degree of sideeffects. Clofibrate and Gemfibrozil have the following side effects:nausea, abdominal discomfort, gastrointestinal discomfort, myalgiaassociated with increased plasma levels of creatine phosphokinase. Inaddition, hepatic dysfunction and bone marrow depression are toxiceffects of the drugs. Patients taking clofibrate are reported to havedecreased libido and breast tenderness. Brittle hair and alopecia havealso been reported. Questran and colestipol have the following sideeffects: the most common complaints are constipation and bloatingsensation; some times heartburn and diarrhea also are reported. Neomycinhas high toxicity. Even at low doses, nausea, abdominal cramps, diarrheaand malabsorption have been reported.

As mentioned above, so far there is no effective and safe drug whichwould decrease high cholesterol and triglyceride levels in the plasma ofpatients and without side effects.

Few drugs have been shown to decrease platelet aggregation. For example,Aspirin has been shown to decrease the incidence of transient ischemicattacks in men and has been used as prophylactic agent for this purpose.Aspirin has the same drawbacks as the above drugs decreasing cholesteroland triglyceride. Aspirin taken at the usual dosage causes the main sideeffect of gastric intolerance. With higher doses, patients mayexperience tinnitus, decreased hearing and vertigo. The adverse effectsof aspirin on renal function and on the liver has been reported.Hypersensitivity reactions may occur in patients with asthma and nasalpolyps and may be associated with bronchoconstriction and shock. Aspirinhas serious side effects on patients with hemophilia.

In tradition, Curcuma longa L., Angelica sinesis diels and Artemisiacapillaris thunb as natural herbs have been used in clinic fordecreasing levels of hyperlipidemia and anti-aggregating activity ofplatelet.

    ______________________________________                                        Reference:                                                                    ______________________________________                                        (1) Chung King Medical College Acta                                                                        1:88     1979                                    (2) New Medical Journal     11:35     1977                                    (3) Journal of Traditional Chinese Medicine                                                               21:39     1980                                    (4) Journal of Traditional Chinese Medicine                                                                23:762   1982                                    ______________________________________                                    

SUMMARY OF THE INVENTION

It is an object of the present invention to provide pharmaceuticalcompounds and composition which are safe and highly effective forreducing hyperlipidemia and inhibiting platelet aggregation.

These objects and other objects will become apparent hereinafter uponreading the detailed description of the invention. The present inventionresides, briefly stated, in new compositions comprising a mixture of thefollowing active ingredients:

(1) Aurapten or Scopatone extracted from peels seeds or young plants ofCitrus aurantium L, Citrus spp, Poncirus trifoliata raf or Artemisiacapillaris Thunb.

(2) Curcumin extracted from Curcuma longa L, Curcuma aromatica salisb orCurcuma zedoaria roscoe.

(3) The Ferulic Acid extracted from Levisticum officinale koch, Angelicasinensis Diels, or Angelica spp.

(4) Yejuhua-flavonoid extracted from Matricaria chamomilla L, Tagetesminuta L, Tagetes Patula L or Chrysanthemum indicum L.

(5) Additive ingredient: Fish oil concentrate.

For the sake of convenience, compositions comprising mixtures of theabove extracts will hereinafter be referred to as "PHP".

The following herbs: Levisticum officinale koch, Matricaria chamomillaL, Citrus aurantium L, Citrus spp. Curcuma longa L, Angelica spp,Tagetes minuta L and Tagetes patula L are recognized by FDA of U.S. assafe for human consumption.

DETAILED DESCRIPTION

Among all the diseases today in the entire world, coronary arterydisease and cerebrovascular accident bring about the highest mortality.Coronary artery disease and cerebrovascular accident are believed toinvolve following mechanisms: hyperlipidemia and highplatelet-aggregation.

Obviously, reducing hyperlipidemia and inhibiting platelet aggregationare both very important. Consequently the aim of the present inventionis to provide for new and safe therapeutic compounds which will fulfillpractical needs more effectively than previously known drugs in that thecompounds not only reduce hyperlipidemia but also inhibit plateletaggregation at the same time.

Therefore PHP is a bifunctional drug. So far some drugs used forreducing hyperlipidemia and other drugs used for inhibiting plateletaggregation only. Meanwhile all the mentioned drugs have a certaindegree side effect.

Coronary artery disease and cerebrovascular accident are caused byhyperlipidemia and high platelet-aggregation. Therefore PHP caneffectively treat and prevent coronary artery disease andcerebrovascular accident.

PHP be administered to patients in the form of capsules containing apowdered mixture of the active ingredients in appropriate proportions.Alternatively, tablets can be prepared comprising the active ingredientsand pharmaceutically acceptable binders, excipients, lubricants,sweeteners and coatings. A syrup or elixir may be prepared by dissolvingPHP in alcohol and water together with suitable preservatives,sweeteners, dyes and flavoring agents. Ampules or vials for injectionmay likewise be prepared, with the PHP prepared as for oraladministration and after being purified through furtherrecrystallization and sterilization and with the addition thereto ofdistilled water and other suitable solvents and additives known in thepharmaceutical art.

The PHP dosage units prepared according to the invention can beadministered to patients. PHP is nontoxic.

The compositions of the present invention all include as their activecomponent PHP, which as indicated previously, consists of a mixture offour plant extracts: Aurapten or Scoparone, Curcumin, Yejuhna-flavonoid,Ferulic acid and additive ingredient: fish oil concentrate.

Aurapten and Scoparone have the following structural formula: ##STR1##When R₁ =CH₃ O, R₂ =CH₃ O, the compound is scoparone. When R₁ =H##STR2## the compound is aurapten.

Curcumin has the following structural formula: ##STR3##

Ferulic acid has following structural formula: ##STR4##

The fish oil concentrate is derived from the fish body (include scalesof a fish). Fish oil is rich in Eicosapentaenoic acid (EPA) andDocosahexaenoic acid (DHA).

EPA is the precursors in the synthesis of prostaglandins which may helpantiplatelet-aggregating activity and help prevent thrombosis.

The following specific examples will provide detailed illustrations ofmethods of producing PHP according to the present invention andpharmaceutical dosage units containing PHP. Moreover, examples will begiven for pharmaceutical testing performed with PHP which demonstratesits effectiveness in reducing hyperlipidemia and reducingplatelet-aggregation. These examples are not intended, however, to limitor restrict the scope of the invention in any way, and should not beconstrued as providing conditions, parameters, reagents, or startingmaterials which must be utilized exclusively in order to practice thepresent invention.

EXAMPLE 1 Scoparone Extracted from Young Plants of Artemisia capillarisThunb:

Young plants of Artemisia Capillaris Thunb were dried and powdered. Onekilogram (kg) of the powder was dipped in 3 liters of 95% ethanol forabout 12 hours at 50° C. The resultant ethanol extract was concentratedunder reduced pressure by distillation. A residue was recovered anddissolved in 300 ml 50° C.-water. The resulting aqueous solution waswashed twice with chloroform. The solution was heated to remove thechloroform. Then, the solution was extracted twice with ethyl acetate.Ethyl acetate extract was removed and dried by sodium sulfate. Ethylacetate was then recovered by reduced pressure distillation and theresidue was dissolved in warm-methanol. The methanol solution wasconcentrated, and was allowed to stand at room temperature overnight.White or white-yellow crystal line were formed. The crystal line wererecrystallized from methanol and water. They were dried under vaccum andfound to have a melting point of 145°-146° C. The resulting white orwhite-yellow crystal line was the final product: scoparone.

EXAMPLE 2 Extraction of Curcumin

Rhizome of Curcuma Longa L., Curcuma aromatica Salisb or CurcumaZedoaria roscoe was dried and powdered. One kilogram of the powder wassoaked in 5 liters of 95% ethanol for about 12 hours at roomtemperature. The resulting ethanol extract was filtered. Ethanol wasthen recovered under reduced pressure distillation. A residue wasdissolved in 300 ml of 1N NaOH, then the resulting solution was adjustedto PH 7 with 1N HCl, and a light yellow precipitate was formed. Theprecipitate was dissolved in 300 ml of 95% ethanol and the lastprocedure was repeated to form a light yellow precipitate again. Thelight yellow precipitate was washed with water, acetone and ethersuccessively. The final light yellow powder was dried under vacuum andwas found to have a melting point of 183° C.

EXAMPLE 3 Ferulic Acid Extracted from Levisticum Officinale Koch orAngelica sinesis diels

1 kg of dried powder of Levisticum Officinale Koch or Angelica sinesisDiels is extracted with 5000 ml of boiling water for 1 hour. Theextraction is repeated twice. Extracts are combined and distilled to1000 ml by reduced pressure distillation. Add 2000 ml 95% ethanol to1000 ml the residue with stir. Set 24 hours. Ethanol mixture is filteredand the filtrate is concentrated to syrup under reduced pressuredistillation. Add 300 g silica gel to the syrup mixture is cooled to theroom temperature and dried at 60° C. Dried powders are washed with 95%ethanol in a percolator until ethanol becomes light color. Ethanolwashes are combined and distilled under reduced pressure whereby ethanolis recovered and a still residue is obtained. This still residue isdissolved in 200 ml of distilled water. This aqueous solution is addedto a packed with polyamide resin. The column is washed with distilledwater, and followed by 50% ethanol. The 50% ethanol is collected anddistilled under reduced pressure. The residue is dissolved in 100 ml ofmethanol. This methanol solution is added to 5 g silica gel. Dried. Themethanol solution is passed through a column (4×22 cm). Wash column bybenzolacetone (7:3). The benzol-acetone is combined and distilled underreduced pressure. Crystals are obtained. Crystals are recrystallizedagain from chloform-methanol (1:1). White crystals are obtained. Meltingpoint is 169.5°-171° C.

EXAMPLE 4 Extraction of Yejuhua-flavonoid from Chrysanthemum indicum L

Plants or flowers of Chrysanthemum indicum L were dried and powdered. 1kg of the powder was extracted in 2 liters of 95% ethanol for about 24hours at room temperature. The solution was filtered and the extractfiltrate saved. 2 liters of 95% ethanol was added to residue andrefluxed in water bath and refluxing for 6 hours. The refluxed ethanolwas cooled and filtered and the filtrate combined with the extractfiltrate. Ethanol was then recovered by reduced pressure distillationand a residue was saved. 1000 ml of ethyl acetate was added to theresidue and refluxed in a water bath for 6 hours. The refluxingprocedure was repeated. Ethyl acetate was then concentrated underreduced pressure distillation. Crystal line were formed. The crystalline were washed with water. Final crystal line were dried under vacuumand was found to have a melting point of about 250° C.

EXAMPLE 5 Preparation of PHP

(1) Fine product of PHP:

Fine PHP prepared according to the present invention consists of about10%-40% (weight percent) scoparone or aurapten, 10%-40% Ferulic acid,5%-30% curcumin, 5%-30% yejuhua-flavonoid and 10%-50% fish oil.Preferred composition weight percent according to the present inventionconsists of about 25% scoparone or aurapten, 25% ferulic acid, 10%curcumin, 10% yejuhua-flavonoid and 30% fish oil.

The dry ingredients of PHP, prepared in accordance with the presentinvention, may be incorporated into tablets, capsules, syrups or otherform by conventional methods.

(2) Crude product of PHP:

Crude PHP is extracted from the above mentioned plants by ethanol.

Proportion of plants extracts and an additive, for example, is asfollows (by weight):

Levisticum officinale Koch or Angelica spp: 21%,

Artemisia capillaris Thunb or Citrus spp: 21%,

Curcuma longa L or C Aromatica Salissb: 14%,

Chrysanthemum indicum L or Matricaria chamomilla L: 14%,

Fish oil: 30%.

Tissues of plant were dried and powdered. 5 liter of distallatory waterwas added 1 kg of dried powder. The mixture was heated to boil andsimmered for one hour. This water extraction was repeated two times.Aqueous extracts are combined and filtered. The filtrate wasconcentrated under reduced pressure to approximately 500 ml. Then 1000ml 95% ethanol was added to the 500 ml aqueous extracts with stirring,which was then distilled and filtered. A residue and a filtrate (A) wereobtained. 1000 ml 90% ethanol was added to the residue with stirring.90% ethanol extraction was repeated twice. The 90% ethanol extracts werecombined. Filtered. Filtrate (B) was obtained. Combined filtrate (A) and(B) was concentrated to a syrup by reduced pressure distillation whileethanol was recovered. The syrup was dried under vacuum and granulatedto final powder. The final powder may be combined with fish oil. Weightof every capsule and table is about 100-200 mg. Crude-PHP is similar tofine-PHP in pharmacological property.

EXAMPLE 6 The influence of PHP on aggregation of platelets

(1) Methods for blood of animal

Aggregation of platelet testing: Rabbit blood was sampled by cardiacpuncture from rabbit with silicon-coated syringe. The blood was mixedwith 3.8% sodium citrate at 9:1 and spun at 1,000 rpm for 6 minutes, 1ml of the platelet-rich plasma was transferred to a silicon-coated 2 mlcell. Mix well and read for transmittance (Ti), with aspectrophotometer. Then 0.02 ml of ADR (10 μM) was added. Stirconstantly and read for transmittance of the platelet-containing-plasmaonce by every one minute and obtain the maximal transmittance (Tm)within 10 minutes. Spin the blood sample at 3000 rpm for 45 minutes andread for transmittance To, of the platelet-poor plasma, the plateletaggregation rate is calculates as follows: ##EQU1##

Results were illustrated by following Table 1

    ______________________________________                                        Aggregation rate of platelet (%)                                              ______________________________________                                        control group  PHP group                                                      (normal saline)                                                                              (1 mg/ml PHP)                                                  50.4 ± 8.1 (*14)                                                                          20.9 ± 1.20 (*14)                                           P < 0.001                                                                     ______________________________________                                    

Table 1 as shown above: the influence of PHP on aggregation of plateletsin vivo. * indicates number of sample.

(2) Methods for blood of humans:

Blood was collected from veins of humans using a needle attached to aplastic disposal syringe. The blood was immediately transferred intosiliconized glass tube containing 0.1 volume of 3.13% sodium citrate.Platelet-rich plasma (PRP) was obtained by centrifugation of the wholeblood at 1000 rpm for 10 min at room temperature. Platelet-poor plasma(PPP) was prepared by centrifugation of the remaining blood at 3000 rpmfor 10 min. Platelet aggregation was performed using in aggregameter at37° C. Human platelet studies were carried out at constant plateletnumber (3×10⁸ /ml). With regards to determination of plateletaggregation, the maximum aggregation induced by adenosine diphosphate(ADP) in a final concentration of 2 μM was obtained by the lighttransmission method. 0.4 ml PRP of each subject was introduced into eachof 24 tubes and divided into 2 groups. Then to the 12 tubes of eachgroup were added 50 μl of saline and 50 μl PHP (0.5 mg/ml) respectively.After incubation of 3 min at 37° C., to each of 12 tubes of each groupwere added 50 μl of 2 μM ADP. A 5 minutes aggregation curve for eachtube was plotted.

Percent inhibition of aggregation by PHP was calculated by: ##EQU2##

Statistical analysis of the results was carried out using Student'st-test for paired data.

    ______________________________________                                                 Rate of aggregation                                                                       Percent inhibition                                                of platelet of aggregation                                           ______________________________________                                        Control    67.5 ± 5.0 --                                                   PHP        20.9 ± 0.9 69.0%                                                P          <0.01                                                              ______________________________________                                    

EXAMPLE 7 The Influence of PHP on Lowering Heperlipidemia

As test animals, 30 male mice weighing of 12-22 grams (g) were used.They were divided into the following three groups. Each group consistsof ten mice.

(A) Standard group

Each animal in standard group is given a daily 0.5 ml of distillatorywater by stomach-tube.

(B) Control group

Each animal in control group is given a daily 0.5 ml ofcholesterol-emulsion. Cholesterol-Emulsion has the following materials:

a. cholesterol: 5 g

b. sodium deoxycholate: 0.5 g

c. lard: 10 g

d. tween: 10 ml

e. propylene glycol: 10 ml

Water was added: 50 ml.

(C) PHP group

Each animal in PHP group is given a daily 0.5 ml of cholesterol-emulsionand about 80 mg/kg of PHP. Mice of three groups were nurtured using astandard diet.

The mice of the above three groups were raised for 10 days. At the endof 10-day test period the animals are weighed and sacrificed, serumcholesterol and triglycerides of liver are determined from blood samplesthaken from the venae cava. The methods are described as following:

Chemical colorimetric methods have been used in the analysis of serumcholesterol.

(D) Reagents and materials

1. Isopropanol, reagent grade.

2. Adsorbent Mixture for the removal of bilirubin, phospholipids,monoglycerides, diglycerides, glucose, and other chromogenic material.Mix well the following materials:

a. Alumina 50-100 mesh, 900 g.

b. Zeolite (Taylor), group and sifted to 20-80 mesh, activated byheating at 110° C. overnight, 50 g.

c. Lloyd's reagent, 50 g.

d. CuSO₄ anhydrous powder, 10 g.

e. Ca(OH)₂ anhydrous, 20 g.

3. Sulfuric Acid, concentrated, reagent grade.

4. Ferric Chloride Color Reagent. Place 500 mg FeCl₃ 6H₂ O in 500 mlvolumetric flask, add glacial acetic acid to the mark, and mix. Thereagent is stable in the dark for 1 year at room temperature.

5. Cholesterol Standard, 200 mg/dl. Dissolve 200.0 mg cholesterol inisopropanol and make up to 100 ml volume.

6. 20×150 mm screw-capped culture tubes with teflon-lined caps.

(E) Procedure

The development and measurement of the color after preparation of theextract has been made by the following methods:

1. Extraction: Pipet 9.5 ml isopropanol into all culture tubes to beused for samples and controls and 9.0 ml isopropanol plus 0.5 ml waterto tubes for standards.

2. Pipet 0.5 ml of serum into the appropriate sample and control tubesand 0.50 ml of cholesterol standard into the standard tubes. Tightlystopper and shake vigorously or mix on a vortex-type mixer for 20seconds.

3. Allow to stand about 20 minutes, add about 2 g of adsorbent mixtureto each tube, and mix thoroughly for 20 seconds. Let stand for 30minutes, and shake vigorously for 5 second every 10 minutes.

4. Centrifuge for 10 minutes at 1100 to 1200 g. Aliquots of the extractcan be used for the determination of cholesterol. Samples with grosslyelevated concentrations can be diluted with isopropanol and re-assayed.

5. Color reaction. Prepare a blank by pipetting 1.0 ml of isopropanolinto a tube. Transfer 1.0 ml of sample, control, and standard extracts,respectively, into appropriately marked tubes.

6. To each tube, add 2 ml FeCl₃ reagent and mix.

7. Add 2 ml concentrated H₂ SO₄ to a tube by allowing the acid to rundown the side of the slanted tube, tightly stopper, and mix by inversion6 times. Then proceed to the next tube.

8. Let color develop for 10 minutes, transfer to a cuvet, and read theabsorbance against the blank at 540 nm.

9. Calculations. Let A_(u) be absorbance of sample and A_(S) theabsorbance of standard; read against the blank. ##EQU3##

    ______________________________________                                                Cholosterol of serum                                                                         Triglyceride of liver                                          (mg/dl)        (mg/100 g)                                             ______________________________________                                        Standard group                                                                          201 ± 15 (10) 12.2 ± 1.8 (10)                                 Control group                                                                           420 ± 20 (10) 27.9 ± 4.5 (10)                                 PHP group 288 ± 18 (10) 12.4 ± 1.9 (10)                                 P         <0.01            <0.001                                             ______________________________________                                    

EXAMPLE 8 Safety of PHP

10% solution of PHP was administered intraperitoneally in mice. Noreactios were observed. The acute LD₅₀ was found to be 1050 mg/kg. Eachdose for an adult is 20 mg. Using 50 kg as the average weight of anadult the dosage is 0.4 mg/kg, therefore it is very safe.

It will thus be shown that there are provided compositions and methodswhich achieve the various objects of the invention, and which are welladapted to meet the conditions of practical use.

As various possible embodiments might be made of the above invention,and as various changes might be made in the embodiments set forth above,it is to be understood that all matters herein described are to beinterpreted as illustrative and not in a limiting sense.

What is claimed as new and desired to be protected by Letters Patent isset forth in the appended claims.
 1. A process for producingyejuhua-flavonoid from a plant selected from the group consisting ofChrysanthemum indicum L., Tagestes patula L. and Tagestes minute L.comprising:a. extracting a powder of the plant with 95% of ethanol for24 hours; b. separating the ethanol extract from the powder; c.concentrating the separated ethanol extract under reduced pressure toyield a residue; d. extracting the residue with ethyl acetate; e.concentrating the resulting ethyl acetate extract under reduced pressureto precipitate crystal line yejuhua-flavonoid; and f. recovering thecrystal line yejuhua-flavonoid.
 2. The process of claim 1 furthercomprising washing said crystal line yejuhua-flavonoid with water anddrying the crystal line yejehua-flavonoid under vacuum.
 3. The processof claim 1 further comprising the steps of:g. extracting the powder ofstep b with 95% of ethanol with reflux; h. recovering a second ethanolextract and combining the same with the ethanol extract of step b.